This proposal seeks to use stable, inducible antisense expression to evaluate folylpolyglutamate synthetase (FPGS) as a cancer chemotherapeutic drug target and to define its role in antifolate resistance. This enzyme catalyzes the addition of five or six glutamyl residues to all folates and classical folate antagonists. As such, it plays a central role in their cellular retention and affinity for target enzymes. For this reason, and because it is avidly expressed in tumor tissue, FPGS continues to attract a great deal of attention as a potential chemotherapeutic drug target. However, to date, the potent inhibitors that have been synthesized are not taken up by tumor cells and are not cytotoxic. For the purpose of testing the hypothesis that FPGS is a target worth further pursuit, we intend to construct an inducible antisense expression system and transfect it into a human leukemia cell line to evaluate the impact of FPGS down-regulation on intracellular folates and cytotoxicity. A critical feature of this study will be use of the radioenzymatic analytical methods, developed during previous phases of this research program, to measure reduced folates and their polyglutamates. FPGS down-regulation results will be compared to direct intracellular inhibition using known inhibitors introduced via artificial membrane permeabilization. The down-regulated system will also provide an excellent model to directly evaluate the relatively widespread proposition that under-expression of FPGS is a folate antagonist resistance mechanism. In addition, in vitro comparison of polyglutamylation between antifolate sensitive and resistant leukemia bone marrow cells will address the clinical relevance of FPGS expression. Overall, results from these experiments should provide a definitive answer to; 1. whether FPGS is a suitable cancer chemotherapeutic target for new drug development, 2. the role of the enzyme in folate antagonist resistance, and 3. the basis for sensitivity of some leukemias, but not others, to the therapeutic effects of folate antagonists.